Glycans on Notch EGF repeats are critical regulators of Notch signaling and thus control the developmental fate of many cell types. Both constitutive and defective Notch signaling lead to disease and cancer. Therefore, it is critically important to determine all factors that contribute to robust Notch signaling. Inactivation of Pofut1, which encodes the protein O-fucosyltransferase that transfers O-fucose to Notch, causes severe disruption of Notch signaling. Pofut1 requires the substrate GDP-fucose that must be transported into the secretory pathway. Mice lacking the known GDP-fucose transporter Slc35c1, and the putative GDP-fucose transporter Slc35c2, exhibit comparatively mild Notch signaling defects, revealing the existence of an additional, novel GDP-fucose transporter essential for Notch signaling. Specific Aim 1 will identify the GDP key-fucose transporter(s) required for Notch signaling in mammals. We will use Slc35c1/Slc35c2 double knockout mutant fibroblasts to isolate the GDP-fucose transporter(s) required for optimal Notch signaling in mammals, define their location in the secretory pathway and establish GDP-fucose transport or transporter chaperone activity. Specific Aim 2 will identify individual and complementary functions of Lfng, Mfng and Rfng glycosyltransferases that transfer N-acetylglucosamine (GlcNAc) to O-fucose on Notch, and thereby regulate Notch signaling. All three are necessary for optimal T cell development. Mice expressing a single Fng gene, all three Fng genes or no Fng genes will be used to reveal structure/function relationships for each Fng in regulating Notch1 signaling and determining T cell fates. This will identify critical EGF repeats of Notch1 that must be modified by O-fucose glycans, and the structure of those O-fucose glycans during T cell development. The third aim will focus on a new modification of Notch EGF repeats - O-GlcNAc. We have recently identified genetic interactions between Drosophila Notch signaling genes and the EGF- specific O-GlcNAc-transferase Eogt, and shown that modification of Notch1 EGF repeats by O-GlcNAc is required for optimal ligand-induced activation of Notch1 in HeLa cells. Specific Aim 3 will determine how O-GlcNAc is important for Notch1 signaling in mammalian cells and in the mouse. The effects of the loss of O-GlcNAc on Notch receptor trafficking to, and stability at, the cell surface, for Notch ligand binding, and for Notch signalig in co-culture assays, will be defined. We will investigate Notch signaling defects during embryogenesis and T and B cell development in Eogt null mice expressing only one copy of Notch1. Four O-GlcNAc sites that are conserved between Drosophila and mammalian Notch will be mutated to identify specific functions for O-GlcNAc. The combined results will reveal important new insights into how Notch signaling is regulated by glycans.